Respiratory syncytial virus with a genomic deficiency complemented in trans

ABSTRACT

The invention relates to pneumoviral virions comprising a viral genome that has a mutation in a gene coding for a protein that is essential for infectivity of the pneumovirus, whereby the mutation causes a virus produced from only the viral genome to lack infectivity, and whereby the virion comprises the protein in a form and in an amount that is required for infectivity of the virion. The invention also relates to methods for producing the pneumoviral virions and for using the virions in the treatment or prevention of pneumoviral infection and disease. A preferred pneumoviral virion is a virion of Respiratory Syncytial Virus in which preferably the gene for the G attachment protein is inactivated and complemented in trans.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.14/815,131, filed Jul. 31, 2015, which is a Divisional of U.S.application Ser. No. 10/583,800, now U.S. Pat. No. 9,107,939, which isthe U.S. National Stage of International Patent Application No.PCT/NL2004/000911, filed Dec. 24, 2004, and which claims benefit toInternational Patent Application No. PCT/NL2003/000930 filed Dec. 24,2003, the entire disclosures of which are incorporated herein byreference.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirely. Said ASCII copy, created on Jan. 5, 2018 isnamed 069818-6328SequenceListing.txt and is 32 KB.

FIELD OF THE INVENTION

The present invention relates to the field of vaccination, and morespecifically to vaccines against disease caused by pneumoviruses such ase.g. Respiratory Syncytial Virus (RSV). The invention pertains RSVvirions carrying an RSV genome in which a gene that is essential forinfectivity has been inactivated, while the corresponding wild typegene-product is complemented in trans to the virion. The inventionfurther relates to methods for the production of such RSV virions and totheir use in vaccines and methods for vaccination against pneumoviruses.

BACKGROUND OF THE INVENTION

Human respiratory Syncytial virus is classified in the genusPneumovirus, family Paramyxoviruses. It is a major cause of severe lowerrespiratory tract disease in infants, the elderly and inimmunocompromised individuals. It is also an important factor in upperrespiratory tract disease in older children and adults. Currently thereis no effective h-RSV vaccine available in the art.

RSV is an enveloped RNA virus that expresses two major antigens at itssurface: the attachment protein G and the fusion protein F. Bothproteins appear to invoke protective antibodies. G is the determinant ofthe two known h-RSV subgroups A and B. Antigenic differences can befound within the two groups. The G protein shows a high degree ofvariation with only 53% amino acid homology between groups A and B andup to 20% differences in G protein sequences within group A (Mufson1988, Cane 1991).

Passive immunisation with RSV-enriched immunoglobulin (Respigam) orsynthetic humanised monoclonal antibodies against F (Palivizumab) iscurrently used to treat and protect neonates of certain predispositions(e.g. premature birth) against RSV infection (Robinson 2000, Greenough2000). RSV pathology has two major aspects: cell damage caused by thevirus itself and tissue damage caused by the overreacting immune system.The latter is a highly complicating factor in vaccine design.

RSV infections are seasonal, limited to the winter period and peak inthe Northern Hemisphere around the end of the year. RSV infects everychild before the age of two, in many cases twice. Older individuals onaverage are infected every other year, depending on the setting; peoplein close contact with infants and young children have a 50% risk. Thevirus spreads by close contact, in droplets or through contaminatedsurfaces. RSV is not efficiently spread through aerosols; the virusparticles are relatively unstable. Internal spread of the virus from theupper respiratory tract (URT) to the lower respiratory tract (LRT)occurs predominantly by inhalation of virus particles produced in theURT epithelium during primary infection. Spread through syncytiumformation (one of the pathological properties of the virus, which gaveit its name) can not be ruled out and may play a secondary role in LRTinfection.

In general, RSV pathology starts in the URT; the port of entry is thenose and to a lesser extent the eves not the mouth. When restricted toURT tissues, disease is limited to common cold, although in adultssometimes severe. However, when the virus can reach the LRT,bronchiolitis and pneumonia can ensue in unprotected individuals. Inyoung infants, this can be life threatening, approx. 1/100 will requirehospitalisation and mechanical ventilation, out of these 1% may die. Inthe elderly, RSV-induced LRT disease is a major cause ofhospitalisation; it is suspected that RSV causes 25% of flu-likediseases.

The immune response to RSV is complex. In general, exposure to h-RSVwill build up a response that protects against LRT disease. Thisresponse wanes with older age, causing the higher susceptibility to RSVof the older population. Effective long lasting protection against URTdisease appears not feasible: re-infection is very common, even withinthe same season and this is not caused by viral variation. Protectionagainst RSV infection involves antibodies against viral proteins F and Gcirculating in the blood, which can prevent LRT disease. URT infectioncan be controlled by mucosal antibodies against F and G, but these havea limited life span. CD8+ T cells against as yet unidentified viralproteins are required to clear the virus from infected tissues, but theyappear to be short-lived or inefficiently recruited from theirreservoirs. Most likely, this is caused by RSV-expressed factors,possibly encoded in the G gene (Srikiatkhachorn, 1997a).

An important aspect of RSV disease is immune enhancement of pathology.In limited cases the cellular immune response can exacerbate RSV diseaseby the action of cytokines on infected tissues released from excessivelyattracted granulocytes. Host predisposition is involved in thisreaction, but possibly also the timing of first RSV infection afterbirth. Unfortunately, early vaccine trials with formalin-inactivated RSVshowed that in these vaccination settings immune enhanced pathology uponwt infection was prevalent (Kim 1969). Factors contained in RSV appearto be responsible for this phenomenon and were apparently released byformalin treatment. In the 40 years since then, it was gradually shownthat the viral G protein is the predominant mediator of these problems,but the mechanism remains unclear (Srikiatkhachorn 1997b). In any case,vaccination with a G protein out of the context of the virion (i.e. ininactivated virus preparations, as expression product not properlyembedded in a membrane or in the form of peptides) seems to be causingimmune enhancement in model systems. Thus, although G contributes tosome extent to RSV immunity, its properties also complicate vaccinedesign.

Initial live RSV vaccine candidates included cold passaged ortemperature-sensitive mutants. The former have been attenuated byculturing at decreasing temperature, leading to dependency on lowtemperatures for growth, whereas the latter mutants have been madedependent on a specific, usually higher temperature for replication bychemical or radiation mutagenesis. These live virus vaccine candidatesappeared to be either under- or overattenuated (Crowe 1998).

Subunit vaccine candidates are derived from either the RSV-F or the Gprotein, being the main targets for neutralising antibodies. A candidatesubunit vaccine, PFP2, purified F protein, is sate in RSV-seropositivepatients, but it did not provide full protection against LRT infectionand associated disease (Gonzalez 2000). Another subunit vaccine approachis BBG2Na, which consists of a polypeptide, comprising amino acid130-230 of h-RSV-G, fused to the albumin-binding domain of streptococcalG protein (Power 1997). BBG2Na induces a T helper type 2 response inneonatal mice, and does not elicit lung immunopathology (Siegrist 1999).There is no data yet on protection. The use of new adjuvants for abalanced humoral and cellular immune response are currently widerinvestigation in animal models (Plotnicky 2003).

The use of plasmid-DNA vectors encoding RSV-F and G antigens as vaccinecandidates has been studied in animal models. These vaccines induceprotective responses in rodents (Li 2000), but in one study RSV-F DNAvaccine candidate immunised mice developed a slightly enhanced pulmonaryinflammatory response following challenge with wt virus (Bembridge2000). The feasibility of the use of plasmid DNA vaccines in humans isnot yet known and it will likely take at least 15 years before thisapproach is sufficiently studied and—more importantly—accepted,particularly for neonates. Candidate vaccines based on vector deliverysystems are constructed of live recombinant vectors expressing RSVproteins. For example, recombinant vaccinia virus expressing RSV-F and Gprovided protection in mice, but lacked this effect in chimpanzees(Collins 1990). The question is whether these systems are safe (notablyvaccinia virus) and feasible in the light of existing (maternal)antibodies against poxviruses in the community and the main target groupbeing neonates.

Several vaccine candidates are based on recombinant live RSV, generatedby reverse genetics. One line of study focuses on attenuating theseviruses by introducing the individual or combined mutations responsiblefor cold-adaptation and temperature-sensitivity into the recombinantvirus. None of these vaccine candidates were usable, because of eitherover- or underattenuation. Another line of study focuses on deletion ofone or more viral non-structural genes. Limited data are available onthe behaviour of these viruses in model systems (Jin 2003).

An alternative approach to RSV vaccine development is the use of bovineRSV. A chimeric bovine RSV with either the human F protein alone or boththe human F and G protein was evaluated for its efficacy in chimpanzees.This vaccine candidate was restricted in replication to such a degreethat animals were not protected after wild type h-RSV challenge(Buchholtz 2000).

Thus, currently there is no effective h-RSV vaccine available in theart. All RSV vaccine candidates that have been tested in animal modelsare unusable in humans. There is thus a long felt need in the art forRSV vaccines that are both effective and safe and it is an object of thepresent invention to provide for such vaccines.

DESCRIPTION OF THE INVENTION Definitions

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

The term “virion” as used herein refers to a virus particle thatcontains the nucleocapsid protein, the viral genome and the replicasecomplex in a lipid envelop that contains the viral structuralglycoproteins.

The terms “infectivity of a virus”, “infectious virus”, “infectiousvirus particle” or “infectious virion” denote viruses, virus particlesor virions that are capable of entering suitable host cells andinitiating a virus replication cycle, whether or not this leads to theproduction new virus that is infectious.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention relates to a virion of apneumovirus. The virion comprises a viral genome that has a mutation ina gene coding for a protein that is essential for infectivity of thepneumovirus, whereby the mutation causes a virus produced from only theviral genome to lack infectivity, and whereby the virion comprises theprotein in a form and in an amount that is required for infectivity ofthe virion.

The pneumovirus preferably is a Respiratory Syncytial Virus (RSV), morepreferably a human or bovine RSV. The human RSV may either be a subgroupA or B virus, and preferably is a clinical isolate, more preferably anisolate that has not been extensively passaged in vitro (preferablypassaged less than 10, 8, 6 or 5 times as described in the Examples).Therefore, any RSV strain or isolate may be used in the context of thepresent invention, whereby is understood that the invention is onlyexemplified by means of the particular human RSV isolate 98-25147-X,referred to as RSV isolate X. Further preferred is that virus is arecent clinical isolate whereby recent is defined as being firstisolated less than 10, 8, 6, 4, 3, or 2 years ago. It will be understoodthat although the nucleotide sequences in the virion do not need tocorrespond to those of the recent isolate, preferably, the amino acidsequences of the proteins present in the virion of the invention areidentical to the proteins as they occur in a recent clinical isolate.

The viral genome comprises at least one mutation in at least one viralgene coding for a protein that is essential for infectivity of thepneumovirus, whereby the infectivity of the virus is as defined above.Thus, the protein that is essential for infectivity of the pneumovirusis a protein that is essential for the capability of the virion of theinvention to enter a suitable host cell and initiate a viral replicationcycle, whereby the replication cycle does not necessarily lead to theproduction of new infectious virions. In preferred virions of theinvention the mutation causes the virion to lack infectivity in vivo,i.e. in a suitable host organism, whereby the virions may still beinfectious for suitable host cells cultured in vitro.

In a preferred virion of the invention, the mutated gene that codes fora protein essential for infectivity of the pneumovirus, is a gene, whichcodes for a structural protein of the virus. A structural protein of apneumovirus is herein understood to be a protein that is present invirions of wild-type infectious virus. Preferred genes coding forstructural proteins to be mutated in the virions of the invention arethe genes coding for the attachment protein G and/or the fusion proteinF, whereby the G protein is most preferred. Deletion and/or functionalinactivation the gene coding for G protein serves several purposes andprevents a number of problems and complications of current RSV vaccinecandidates. One purpose is vaccine safety: RSV without G protein ishighly attenuated in its host (Karron 1997, Schmidt 2002) because itwill not be able to efficiently infect host cells. One complication isthat the G protein is strongly implicated in causing undesiredimmunological responses, including enhanced immune pathology (Alwan1993, Srikiatkhachorn 1997b)) and possible skewing of the immune systemtowards an allergy (and asthma-) prone state under certain geneticpredispositions (Openshaw 2003, Peebles 2003). This will be prevented bydeletion or inactivation of the G gene. A pneumoviral virion of theinvention comprising a viral genome that has an inactivating mutation inthe gene coding for a G attachment protein, and comprising the Gattachment protein in a form and in an amount that is required forinfectivity of the virion is referred to as a “ΔG+G” (pneumo)virus orvirion. Similarly, the virion that has the inactivating mutation in thegene coding for a G attachment protein, but which is not complemented intrans with a functional amount of G protein is referred to as a “ΔG”(pneumo)virus or virion.

The pneumoviral virions of the invention are thus transiently andfunctionally reconstituted with an externally encoded protein that isessential for infection. Preferably the externally encoded protein thatis essential for infection is the attachment protein G and/or the fusionprotein F, whereby the G protein is most preferred. Preferably theexternally encoded protein that is essential for infection is of thesame viral subgroup (A or B) as the genome that is present in thevirion. More preferably the externally encoded protein that is essentialfor infection is homologous to the genome that is present in the virion,whereby is meant that the protein has the same amino acid sequence asthe amino acid sequence that was encoded in the genome of the virusprior to its inactivation. Alternatively, this may mean that theexternally encoded protein has the same amino acid sequence as presentin a wild type virion of which the amino acid sequences with one or moreinternally encodes proteins have 100% identity with their counter partin the virion of the invention.

In the virions of the invention, the mutation in the gene of theessential structural protein is a mutation that causes the virusproduced from only the viral genome to lack the protein or to express abiologically inactivated protein. Production of virus from only theviral genome is understood to mean virus produced exclusively from theviral genome as present in the virions and in the absence of any codingsequence complementing the viral genome in trans. The viral genome aspresent in the virions is thus incapable of directing expression of theessential structural protein. This may be achieved in various ways knownto the skilled person, including e.g. inactivation of the translationinitiation codon, introduction of stop codons near the N-terminus of theencoded protein, one or more frame-shift mutations, deletion of one ormore fragments from the gene. Preferably the gene is inactivated bydeletion of at least 10, 20, 50, 75. 90 or 95% of the sequence codingfor the essential structural protein. Most preferred is however, avirion in which the mutation comprises deletion of the (entire) sequencecoding for the protein.

Explicitly included in the invention are virions in which more than onemutation is present. In particular, more than one viral protein-codinggene may comprise mutations that inactivate or alter the function of theprotein in question, or which cause the protein to lack from the virionsas described above. E.g. the cold-passaged or heat-sensitive mutationsas known in the art may be combined with inactivation of the essentialstructural proteins as disclosed in the invention above.

Clearing of pneumoviruses like RSV from the infected host organismsrequires proper cellular immunity, which will not be effectively mountedwithout infection of epithelial cells by the virus. However, the mutantpneumoviruses of the invention lack genetic information for a proteinthat is essential for infection of host cells in vivo. Therefore thepresent invention discloses methods for the production of the mutantpneumoviruses, which include replication of mutant pneumoviruses incells that complement (in trans) for the absence of the protein that isessential for infection.

In another aspect the invention thus pertains to a method for producingthe above defined mutant pneumoviral virions. The method is a method forproducing pneumoviral virions, whereby the virions comprise a viralgenome that has a mutation in a gene coding for a protein that isessential for (in vivo) infectivity of the pneumovirus, whereby themutation causes a virus produced from only the viral genome to lackinfectivity, and whereby the virion comprises the protein in a form andin an amount that is required for infectivity of the virion. The methodcomprises the steps of: (a) infecting a culture of a first host cellwith a pneumovirus comprising a viral genome that has a mutation asdefined above, whereby the host cell comprises an expression vectorwhich directs expression, either transiently or constitutively, in thehost cell of the protein in a form and in an amount that is required forinfectivity of the virion; and, (b) recovery of the virions from theinfected host cell culture. Recovery of virions from the infected hostcell culture may include either or both recovery from the culture mediumas well as recovery from the cells.

The first host cell may be any host cell in which the pneumovirus iscapable of replication, with or without the simultaneous expression intrans of the protein that is required for infectivity of the virion.Suitable host cells for this purpose are e.g. African green monkeykidney cell cultures (such as e.g. Vero, ECACC lot 10-87, 134^(th)passage, 1990, EMEA approved).

In a preferred method of the invention, the pneumovirus that is used toinfect the culture of a first host cell culture, is produced in a methodcomprising the steps of: (a) providing to a second host cell one or moreexpression vectors which direct expression in the host cell of: (i) aviral genomic RNA that has a mutation in a gene coding for a proteinthat is essential for (in vivo) infectivity of the pneumovirus, wherebythe mutation causes a virus produced from only the viral genome to lackinfectivity; and, (ii) a pneumoviral polymerase enzyme complex andoptionally one or more further viral proteins; and, (b) culturing thesecond host cell whereby the virions are produced. In a preferredmethod, the virions produced by the second host cell are amplified byone or more further cellular infection steps employing host cells whichare the same or different from the second host cell.

The second host cell may be any host cell in which the pneumovirus iscapable of replication, with or without the simultaneous expression intrans of the protein that is required for infectivity of the virion.Suitable host cells for this purpose are e.g. African green monkeykidney cell cultures (such as e.g. Vero, ECACC lot 10-87, 134^(th)passage, 1990, EMEA approved), or Hep-2 cells. The second host cell maybe the same as or different from the first host cell.

In the methods of the invention, the viral genomic RNA is transcribedfrom a viral DNA copy that is under the control of a bacteriophageDNA-dependent RNA polymerase promoter and whereby the (second) host cellis provided with an expression vector which directs expression in thehost cell of the bacteriophage DNA-dependent RNA polymerase. Preferably,the bacteriophage DNA-dependent RNA polymerase is a T7, T3 or SP6polymerase.

The pneumoviral polymerase enzyme complex that is expressed from one ormore expression vector(s) in the second host cell at least includes theL, P, N proteins expressed from their corresponding genes or cDNA's inthe expression vector(s). For improved efficiency of viral assembly andpackaging of the naked viral genomic RNA, optionally, one or morefurther viral proteins are expressed in the second host cells. Preferredviral proteins for this purpose include the viral matrix membraneproteins of which the M2-1 protein is particularly preferred. The L, P,N, M2-1, G or F proteins are preferably derived from the viral genome ofthe viral isolate which is introduced and expressed in the host cell,but alternatively also homologous proteins from other heterologous viralor non viral sources may be used.

The skilled person will appreciate that a wide variety of expressionvectors and regulatory sequences (such as promoters) are available inthe art for expression of the viral genomic RNA, the DNA-dependent RNApolymerase, pneumoviral polymerase enzyme complex and optional furtherviral proteins, as well as the essential structural protein, in thefirst and/or second host cells (see e.g. Sambrook and Russell (2001)“Molecular Cloning: A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, New York).

For reverse genetics of RNA viruses, i.e. expression of a recombinantRNA virus such as the virions of the present invention, a cDNA copy ofthe viral genomic RNA is cloned into plasmids and is placed under thecontrol of sequences that will allow synthesis of RNA from the DNA undercertain conditions. Generally, the promoter sequence for bacteriophageRNA polymerase (e.g. the T7 RNA polymerase) is placed upstream of theDNA copy of the RNA genome, while an appropriate terminator for the RNApolymerase is placed downstream of the genome. Self-cleaving ribozymesequences are placed upstream of the terminator sequences, to allowsynthesis of RNA with the correct terminal nucleotides. Correct terminalsequences are generally required to rescue virus from the synthetic RNA.For non-segmented negative strand RNA viruses, co-expression of thepolymerase enzyme complex (N, P and L proteins for Paramyxoviruses)along with the genomic or anti-genomic RNA is required to obtainrecombinant virus (reviewed by Neumann 2002 and exemplified in theExamples herein).

Other preferred methods may comprise the further step of isolatingand/or purifying the virions of the invention and/or formulating thesevirions into pharmaceutical compositions. Methods for isolating and/orpurifying virions are well known to the skilled virologist. Such methodse.g. include various centrifugation techniques (e.g. differential ordensity centrifugation), or chromatographic techniques. A method forformulating the virions of the invention into a pharmaceuticalcomposition at least comprises the step of mixing the virions with apharmaceutically acceptable carrier as defined below.

In a further aspect the invention relates to a composition comprising avirion as defined above or obtainable in a method as defined above, anda pharmaceutically acceptable carrier. The composition preferably is apharmaceutical composition that is preferably suitable for use as avaccine, i.e. the composition preferably is a vaccine.

In a yet another aspect the invention provides for a pharmaceuticalpreparation comprising as active ingredient a virion according to theinvention, and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable stabilising agents, osmotic agents, buffering agents,dispersing agents, and the like may also be incorporated into thepharmaceutical compositions. The preferred form depends on the intendedmode of administration and therapeutic application. The pharmaceuticalcarrier can be any compatible, non-toxic substance suitable to deliverthe reconstituted viral membranes to the patient. Pharmaceuticallyacceptable carriers for intranasal delivery are exemplified by water,buffered saline solutions, glycerin, polysorbate 20, cremophor EL, andan aqueous mixture of caprylic/capric glyceride, and may be buffered toprovide a neutral pH environment.

For administration by inhalation, the pharmaceutical compositions of thepresent invention are conveniently delivered in the form of an aerosolspray from pressurised packs or a nebuliser, wherein the virions arepresent in a carrier as described for intranasal delivery but with theuse of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurised aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.

Methods for preparing intranasal or inhalant compositions are well knownin the art and described in more detail in various sources, including,for example, Remington's Pharmaceutical Science (15th ed., MackPublishing, Easton, Pa., 1980) (incorporated by reference in itsentirety for all purposes). The virions may thus be formulated as theactive component in any preparation for vaccination, which may furthere.g. include carriers, adjuvants, stabilisers, solubilisers,preservatives and other excipients known in the art, to allow or to aidefficient administration of the preparation for vaccination toindividuals, preferably human and live stock or farm animals (such ascows, pigs, horses, goats, sheep).

In a further aspect, the invention relates to a method for vaccinationagainst, or for prophylaxis or therapy (prevention or treatment) of anpneumoviral infection by administration of a therapeutically orprophylactically effective amount of (a pharmaceutical compositioncomprising) the virions of the invention as defined above, or obtainableas defined above, to a subject in need of prophylaxis or therapy.Preferably, the virions are administered intranasally.

The invention similarly relates to virions of the invention as definedabove, or obtainable as defined above, for use as a medicament,preferably a medicament for vaccination against, or for prophylaxis ortherapy of a pneumoviral infection. The invention further relates to theuse of the virions of the invention in the manufacture of a medicamentfor vaccination against, or for prophylaxis or therapy of a pneumoviraldisease or infection. Preferably the medicament is a preparation forintranasal administration.

The compositions comprising the virions of the invention for vaccinationare preferably administered intranasally to appropriate hosts. In oneembodiment, calves are to be protected from b-RSV infections. In yetanother embodiment, humans, of which preferably infants and elderly orimmune compromised individuals are protected from h-RSV infections.Formulations preferably comprise formulations suitable foradministration as intranasal drops or spray, preferably a nasal spray.The ΔG+G-pneumoviral particles in the composition will infect epithelialcells of the upper respiratory tract only once because the secondgeneration virions produced from the initially infected URT epithelialcells lack the G attachment protein for Which the coding sequence hasbeen removed from the genome. These ΔG-virions are thereforenon-infectious in vivo in host organisms. However, the initial singlecycle of infection allows for the development of appropriate cellularimmunity—that is a response capable of clearing wild-type virusinfection—to be mounted against pneumovirus, or RSV in particular, whileprotective antibodies against F—i.e. antibodies that will prevent lowerrespiratory tract infection—will be elicited by the vaccine and thenon-infectious progeny. Anti-F antibodies are effective in limiting RSVinfection, as is shown by the effectiveness of Palivimuzab treatment,which is a humanised monoclonal antibody against F. This is the basis ofthe efficacy of the recombinant live attenuated pneumoviral vaccines ofthe invention. These live viral vaccines solves a number of problemsassociated with current pneumovirus vaccine candidates. The presence ofthe G-protein in its natural context in the virion allows for thedevelopment of appropriate cellular immunity whereas the undesirableeffects of immunity against the isolated G protein that is largelyresponsible for immune enhancement of b-RSV and h-RSV pathology incattle and humans respectively, is avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Diagram of construction of pRSVXΔG. Upper line represents RSVisolate X genomic RNA, with genes indicated. Boxes below representRT-PCR products and oligonucleotide duplexes used for construction:Numbers inside boxes indicate the oligonucleotide numbers as listed intable I. Restriction sites introduced for cloning are indicated. Thefinal cloning scheme is indicated below: circles are plasmids and thearrows show the order of cloning.

FIGS. 2A and 2B Alignments showing the differences between RSV isolate Xand pRSVXΔG sequences. Sequences are shown as alignment of genomicsense. For pRSVXΔG only nucleotides differing from RSV isolate X areindicated. Similar sequences are indicated by dots (.) and gaps areindicated by (-). Gene start signals are single underlined, gene stopsignals double underlined, and the genes are indicated in the captions.Boxes outline the restriction enzyme recognition sites resulting fromthe nucleotide changes introduced. The sequences presented in FIGS. 2Aand 2B correspond with the following fragments of SEQ ID NO: 1:

-   -   Intergenic region between L and M2-2: 6722-6792 of SEQ ID NO:1    -   Intergenic region between P and N: 12855-12899 of SEQ ID NO:1    -   Intergenic region between N and NS2: 14081-14127 of SEQ ID NO:1    -   Region of G gene deletion: 9607-9636 and 10532-10605 of SEQ ID        NO:1.

FIG. 3 Identification of sequence markers in RSV RT-PCR amplificationproducts, digestions digests: a) MluI, b) XmaI, c) SexA-I, d) SnaB-I.

FIGS. 4A and 4B Growth curves of RSV isolate X and ΔG-RSV isolate X.Vero (solid lines) and Hep-2 (dashed lines) cells were infected withvirus at MOI=0.1 and incubated at 37° C. At the indicated time pointscells were harvested and CCID50 titres were determined on Vero cells.

TABLES

Table I. Primers used for RT-PCR cloning of RSV isolate X.

Table II. Primers used for cloning of helper plasmids and for plasmidsused for construction of stable cell lines.

Table III. Primers used for diagnostic RT-PCR on RNA from RSV infectedVero cells.

Table IV. Results cotton rat immunization experiments, protectionagainst RSV infection and RSV-induced pathology by ΔG-RSV isolate Ximmunization.

EXAMPLES

The current invention is illustrated by the following non limitingexamples that are merely used to illustrate specific embodiments of theinvention and should not be read as limiting the general scope or anyaspect of the invention.

Example 1 Viral Isolate, Virus Isolation, Propagation and Storage

The basis for the recombinant h-RSV clone is a clinical RSV isolate,obtained from the Leiden University Medical Centre diagnosticlaboratory. This virus, named 98-25147-X, coded after the patient fromwhich it was isolated, was derived from a diagnostic test on Hep-2 cellsin the period 21-24 Dec. 1998. It was later determined to be a subtype Aisolate and is designated RSV isolate X. The virus was passaged 4 timeson Hep-2 cells in T75 bottles in DMEM (Gibco), 10% FCS, pen/strep/gluand subsequently five times on Vero cells in T75 bottles on in DMEM(Gibco), 10% PCS, pen/strep/glu. The resulting RSV isolate X virus wasused as working stock and stored at −135° C. in 25% or 45% sucrose.

Example 2 Construction of RSV-X cDNA Encoding Viral Genome

Total RNA was obtained by phenol-guanidine isothiocyanate extraction(Trizol, Invitrogen) of stock RSV isolate X infected Vero cells. cDNAwas prepared by reverse transcription using Thermoscript (Invitrogen)reverse transcriptase using random hexamer primers. This cDNA was usedas template for PCR using High fidelity Taq polymerase (Invitrogen)using specific primers containing restriction enzyme recognition sites(Table I and sequence listing). Primers were designed based on thepublished sequences of RSV-A2 (Genbank accession no M74568) and RSV-RSS2(Genbank accession no U39662).

PCR products were first cloned individually in different vectors: primerpairs, vectors, restriction enzyme recognition sites and resultingvector name are listed below. RSV021/RSV047: pCAP vector (Roche),bluntly into Mlu N1, pCAP3 (SH/M/P region)

-   RSV018/019: pCAP vector, bluntly into Mlu N1, pCAP2 (G region)-   RSV016/RSV017: PUC21, Mlu I/Bam HI, pUK5 (M2-2/M2-1F region)-   RSV024/RSV025a: PUC21, Bam HI/Afl II, pUK1 (NS2/NS1 region)-   RSV022/RSV023: PUC21, EcoR V, pUK4 (N region)-   RSV014/RSV015: PUC21, Kpn I/Mlu I, pUK2 (L region)

At least two individual clones derived from two independent cDNAtemplates were sequenced; regions containing differences between the twoclones were sequenced on a third clone. If necessary, clones wererepaired using standard molecular biology techniques known by theskilled person. Additional PCR products covering the binding sites ofthe primers used for cloning were obtained and sequenced. The 5′ genomictermini were determined by poly-adenylation of genomic RNA, followed by

RT-PCR with an oligo(d)T containing primer ALG018 (SEQ ID NO: 34):TTAAAAGCTTTTTTTTTTTTTTTTTTTT andan NS1 gene primer RSV126 (SEQ ID NO: 35):AATTCTGCAGGCCCATCTCTAACCAAAGGAGT.This fragment was cloned into pUC21 using Hind III/Pst I. The 3′-end wasdetermined by RACE (rapid amplification of cDNA ends) ligation PCR. Allsequences were assembled to yield the RSV-X consensus sequence (Seq IDNo. 1).

All sequences were confirmed by PCR cycle sequencing using the BigDyeterminator kit (Applied Biosystems) and analysed by an ABI Prism 310genetic analyser.

TABLE I Primers used for RT-PCR cloning of RSV isolate X Template Primerregion name Sequence L RSV014 AATTGGTACCTAATACGACTCACTATAGGGACGAGAAAAAAAGTGTC RSV015 TTAAACGCGTCATCAAACTATTAACTC M2-2/M2-1/F RSV016AATTACGCGTTAAGCATTAGGATTGAGTG RSV017 TTAAGGATCCGCGCGCTATTATTGCAAAAAGCC GRSV018 AATTGCGCGTTTTTAATGACTACTGG T RSV019TAAGGATCCGTACGTTGGGGCAAATGCAAACATGT CC SH/M/P RSV021TTAACCCGGGGCAAATAAAACATCATGG RSV047AATTCGTACGTATTGTTAGTCTTAATATCTTAGTTCA TTGTTATGA N RSV022AATTCCCGGGATTTTTTTTATTAACTCAAAGC RSV023TTAAACCTGGTAAGATGAAAGATGGGGCAAATACAA AAATGGC NS2/NS1 R5V024AATTGGATCCACCAGGTCTCTCCTTAATTTTAAATTA C RSV025aAATTCTTAAGGGACCGCGAGGAGGTGGAGATGCCAT GCCGACCCACGCGAAAAAATGCGTACAAC HDVRRSV026 GTCCGACCTGGGCATCCGAAGGAGGACG RSV027 ACGTCCTCCTTCGGATGCCCAGGTCGHDVR-T7phi RSV028 TCGTCCACTCGGATGGCTAAGGGAATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGC RSV029GGCCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCC CCAAGGGGTTATTCCCTTAGCCATCCGAGTGGACG

Example 3 Construction of ΔG-RSV Isolate X Full Length Plasmid

The full-length cDNA spanning the entire RSV isolate X genome wasassembled by sequential ligation of PCR fragments (FIG. 1). The“trailer” end is preceded by the promoter for the bacteriophage T7polymerase. To generate correct 3′ ends the cDNA “leader” end is fusedto the hepatitis delta virus ribozyme (HDVR), followed by a terminatorof the T7 RNA polymerase transcription (see FIG. 1).

First, two sets of complementary oligomers encoding the HDVR and the T7terminator RSV026/RSV027 oligo's and RSV028/029 oligo's werephosphorylated with T4 DNA kinase, hybridised and ligated into clonepUK1 (containing genes NS1/NS2) via Rsr II/Not I, giving plasmid pUK3.Then, the Xma I/SexA I fragment of clone pUK4 containing N was ligatedinto plasmid pUK3 via Xma I/SexA I. This plasmid (pUK6) contains theregion from the N gene up to the 3′ leader sequence, fused to the HDVRand a T7 terminator.

Secondly, the Xma I/Eco RV fragment of plasmid pCAP3 was inserted inplasmid pUK5 using Xma I and a filled-in Hind III site. This yieldsplasmid pUK8. Subsequently, pUK 8 was digested with BssH II and BsiW I,ends were filled-in with Klenow polymerase and religated. This plasmidcontains the genes M2-2, M2-1, F, SH, M and P and is named pUK9.

To synthesise a low-copy number vector for the RSV isolate X cDNA, twocomplementary oligomers, RSV011 (SEQ ID NO:36):AGCTTGCGGCCGCGTCGACCCGGGACGCGTCGATCGGGTACCAT and RSV012 (SEQ ID NO: 37):CGATGGTACCCGATCGACGCGTCCCGGGTCGACGCGGCCGCA were phosphorylated with T4DNA kinase, hybridised and inserted in the alkaline phosphatase treatedand Cla I/Hind III digested plasmid pACYC184 (New England Biolabs). Theresulting plasmid is named pACYC184-MCS. Subsequently a Mlu I-Knp Ifragment of pUK2 containing the T7 promoter and L gene was inserted,this intermediate plasmid is named pACYC1. Then, the region from the Ngene up to the 3′-leader sequence, including the fused HDVR and T7terminator sequence of pUK6 was added to pACYC1 using Xma I/Not I. Thisgives intermediate plasmid pACYC2. Finally, the Xma I/Mlu I fragment ofpUK9 containing the M2-2, M2-1, F, SH, M and P genes was inserted intopACYC2, yielding plasmid pACYC3, comprising the whole RSV genome ofstrain X lacking the G gene. Sequence analysis of the latter plasmidrevealed a deletion in the HDVR region, which was repaired and theresulting plasmid is named pRSVXΔG.

In addition to construct pRSVXΔG, construct pACYC24 was generated inwhich the genomic RSV isolate X insert is reverse complemented viainverse PCR. From the construct, antigenomic RSV RNA can be synthesised.In pACYC24, the T7 promoter precedes the 3′-leader sequence, whereas theHDVR and T7 terminator are fused to the 5′-trailer sequence.

All restriction enzyme recognition sites used to construct pRSVXΔG arelocated inside the RSV intergenic regions and do not alter codingsequences or affect transcription signals (as shown in FIGS. 2A and 2B).

Example 4 Construction of Helper Plasmids

Helper plasmids expressing several RSV proteins were constructed asfollows. All required genes are derived from lab-strain RSV-A2 (ATCC#VR1302). Virus was plaque-purified on Hep-2 cells and subsequently usedto infect Vero cells. Total RNA was isolated from these cells byphenol-guanidine isothiocyanate extraction (Trizol, Invitrogen) andsubjected to RT-PCR using High Fidelity Taq polymerase (Invitrogen) anda set of primers specific for RSV genes L, P, N and M2-1 respectively(see Table II). PCR products were subsequently cloned into expressionplasmids pcDNA3, pcDNA6 or pCI, using restriction enzyme recognitionsites as indicated in the table II. Clone sequences were confirmed byPCR cycle sequencing using the BigDye terminator kit (AppliedBiosystems) and analysed by an ABI Prism 310 genetic analyser.

TABLE IIPrimers used for cloning of helper plasmids and for plasmids usedfor construction of stable cell lines. Primer Restriction Gene nameSequence sites L RSV045 TTAACTCGAGTTATTCATTATGAAAGTTG xho I RSv046AATTGGTAcCGGGACAAAATGGATCCC Kpn I P RSV043 TTAATCTAGATTGTAACTATATTATAGXba I RSV012a AATTGGATCCGGGGCAAATAAATCATCAT BamH I GG N RSV010AATTGGATCCGGGGCAAATACAAGATGG BamH I C RSV011TTAACTCGAGATTAACTCAAAGCTCTACA Xho I TC M2-1 RSV124AATTGGATCCGGGGCAAATATGTCACGAA BamH I GG RSV125TTAATCTAGATCAGGTAGTATCATTATTTT XbaI TGGC A2-G R5V042TTAATCTAGAAGTAACTACTGGCGTG Xba I RSV004a AATTGGATCCGGGGCAAATACAAACATGTBamH I CCAAAAACAAGGACC RSV151 AATTCCATGGGGTCCAAAACCAAGGACC Nco I AACGA2- RSV033a AAAAGTATACTTAATGTGATTIGTGCTAT Acc I GAM48 AG RSV034TTTTGTATACTGGCAGCTATAATCTCAACT Acc I TCACTTATAATTGC X-G RSV004aAATTGGATCCGGGGCAAATACAAACATGT BamH I CCAAAAACAAGGACC RSV018aAATTTCTAGATTTTTAATGACTACTGG Xba I T7 pol ALG022TTAATCTAGACMACGCGAACGCGAAGT Xba I CC ALG023AATTAAGCTTACCATGGACACGATTAACA Hind III TCGCTAAGAACG

Example 5 Construction of G-Producing Vero Cell-Lines

Cell lines producing RSV-G protein were constructed using severalmethods:

In method 1, the G gene from either RSV-A2 or RSV isolate X, or the Ggene from RSV-A2, in which the internal translation initiation codon hadbeen disabled by modification using primers RSV033 and RSV034, werecloned into expression vector pcDNA3 or pcDNA6 (Invitrogen) using RT-PCRon RNA from RSV-A2 or RSV isolate X infected Vero cells using primers asindicated in Table II. The plasmids were introduced into Vero cellsusing either chemical agents CaCl₂, co-precipitation, liposome-based orelectroporation (Ausubel 1989). Two methods for isolating stable celllines were used. In the first method, 72 hours after transfection, cellswere split using various dilutions into fresh medium containingselective medium, zeocin for pcDNA3 and blasticidin for pcDNA6. Cellswere fed with selective medium every 3-4 days until cell foci wereidentified. Single colonies were picked and transferred in to 96-wellplate, or seeded in various dilutions to obtain single cells in a 96well plate. Antibiotic resistant colonies were tested on expression ofRSV-G by immunostaining techniques or FACS using RSV G-specificantibodies. Colonies expressing G were passaged, and were designated asstable cell lines expressing G. The second method comprises FACS sortingusing RSV-G specific antibodies 72 hours after transfection. RSV-Gexpressing cells were seeded in a serial dilution to obtain single cellsin a 96-well plate and cultured with selective medium. Single cellcolonies were passaged on selective medium and subsequently tested againfor expression of RSV-G, resulting in cell lines expressing RSV-G.

In method 2, the Flp-In system (Invitrogen) is used to produce Verocells with target gene insertion sites at chromosomal positions whichallow different levels of target gene expression. The RSV-G gene,derived from the plasmids from method 1 but with a modification(introduced using primer RSV151: Table II) of the G translationinitiation codon surrounding sequence to allow higher translationlevels, were inserted in each of these cell lines using thesystem-generic method, resulting in Vero cell lines stably expressingdifferent levels of G protein.

In method 3, Vero cells were transiently made to express the G protein,by either transfection with the expression plasmids containing the Ggene from method 1, or by infection with Modified vaccinia virus Ankara(MVA) (Sutter 1992) or fowlpox viruses (Spehner 1990) expressing the Gprotein.

Example 6 Construction of Bacteriophage T7-Polymerase-Producing CellLines

The bacteriophage T7 polymerase gene is PCR amplified from plasmid pPRT7(van Gennip 1997), containing the gene, using primers ALG022 and ALG023(Table II). The PCR product is cloned into pcDNA6b vector, using HindIII/Xba I, yielding plasmid pc6T7pol. Vero cells were transfected usinglipofectamine 2000 as recommended by the manufacturer (Invitrogen). 72hours after transfection cells were split and grown in fresh mediumcontaining blasticidin. Cells were fed fresh medium every 3-4 days andsplit twice to obtain larger culture volumes. 20 days aftertransfection, blasticidin resistant cells were transfected with reporterplasmid pT7-IRES2-EGFP using lipofectamine 2000. For the construction ofplasmid pT7-IRES2-EGFP, first plasmid pT7-EGFP was constructed byinserting via HindIII/BamHI in plasmid p-EGFP-N1 (Clonetech) a set ofcomplementary oligomers encoding for the T7 promoter sequence (ALG32(SEQ ID NO: 38): AGCTAATACGACTCACTATAGGGAGACGCGT and ALG33 (SEQ ID NO:39): GATCACGCGTCTCCCTATAGTGAGTCGTATT). Plasmid pT7-IRES2-EGFP was thenconstructed by cloning the 17-EGFP fragment of plasmid pT7-EGFP intoplasmid p-IRES2-EGFP via XmaI-NotI. Cells expressing EGFP were sorted byFACS and grown in limited dilution to obtain single cell colonies.Single colonies expressing T7 RNA polymerase were tested for stability,grown to larger culture volumes and stored.

Example 7 Method to Produce Recombinant ΔG-RSV Isolate X Virus

Hep-2 cells were cultivated in DMEM+10% FCS (foetal calfserum)+penicillin/streptomycin/glutamine, whereas Vero cells andderivatives thereof are cultivated in M199+5% FCS+pen/strep/glu. Cellswere grown overnight to 80% confluence in 10 mm² dishes at 37° C. ForVero and Hep-2 cells, cells were infected with modified virus Ankara-T7(MVA-T7) (Sutter 1992, Wyatt 1995) or fowlpox-T7 virus (Britton 1996) atMOI=3 (multiplicity of infection 3) and incubated at 32° C. for 60 minprior to transfection, to allow expression of bacteriophage T7polymerase. The cells (Hep-2, Vero or Vero-T7 cells) were washed withOptimem medium (Optimem 1 with glutamax, Invitrogen) and subsequentlytransfected with helper plasmids encoding the N, P, L and M2.1 genes ofRSV and with plasmid pRSVXΔG, using Lipofectamine2000 (Invitrogen) inOptimem (total volume 500 μl). The following amounts of plasmids wereadded: 1.6 μg pRSVXΔG, 1.6 μg pcDNA6-A2-N, 1.2 μg pcDNA3-P, 0.4 μgpcDNA6-A2-L, 0.8 μg pcDNA6-A2-M2.1. After 3-4 hrs of incubation at 32°C., 500 μl of Optimem medium with 2% FCS was added and the cells wereincubated at 32° C. for 3 days. Cells were then scraped and the mixtureof scraped cells and medium containing the rescued virus was used toinfect fresh cultures of Vero or Hep-2 cells grown in DMEM+2%FCS+pen/strep/glu. The latter procedure was repeated for 4-5 times toobtain high titre virus stocks.

Identity of ΔG-RSV isolate X virus was confirmed by RT-PCR on RNAisolated from ΔG-RSV isolate X infected Vero cells and digestion of theobtained products with the unique restriction enzymes whose recognitionsites were introduced into pRSVXΔG (FIGS. 2A and 2B). RSV isolate X wasused as control.

For the identification of sequence markers in RSV, Vero cells wereinfected with RSV isolate X or with ΔG-RSV isolate X with an MOI=0.1. 72hrs after infection, RNA from culture supernatants was isolated and usedas template for RT-PCR. Primers were designed to flank the insertedsequence markers in the recombinant ΔG-RSV isolate X virus. AfterRT-PCR, the obtained products were digested with the appropriaterestriction enzymes. The following digestion products were obtained(FIG. 3):

a) PCR with primer RSV065 (SEQ ID NO: 40) (GTCCATTGTTGGATTTAATC) andRSV093 (SEQ ID NO: 41) (CAAGATAAGAGTGTACAATACTGTC) and digestion withMlu-I yielded the expected fragments of 937 bp for RSV isolate X, and459 and 478 bp for ΔG-RSV isolate X

b) PCR with primers RSV105 (SEQ ID NO: 42) (GTTGGATTGAGAGACACTT) andRSV113 (SEQ ID NO: 43) (AGTATTAGGCAATGCTGC) followed by digestion withXma-I yielded the expected fragments of 880 by for RSV isolate X, and656 and 224 by for ΔG-RSV isolate X

c) PCR with primers RSV112 (SEQ ID NO:44) (CCCAGTGAATTTATGATTAG) andRSV160 (SEQ ID NO:45) (AATTGGATCCATGGACACAACCCACAATGA) and digestionwith SexA-I yielded the expected fragments of 694 bp for RSV isolate X,and 492 and 202 bp for ΔG-RSV isolate X

d) PCR with primers RSV098 (SEQ ID NO:46) (TGGTAGTTCTCTTCTGGCTCG) andRSV114 (SEQ ID NO:47) (ATCCCCAAGTCATTGTTCA) followed by digestion withSnaB-I yielded the expected fragments of 1820 bp for RSV isolate X, and507 and 387 bp for ΔG-RSV isolate X.

Growth characteristics of ΔG-RSV isolate X compared to RSV isolate Xwere determined on Vero and on Hep-2 cells (FIGS. 4A and 4B).

TABLE III Primers used for diagnostic RT-PCR on RNA fromRSV infected Vero cells. Primer name Sequence SEQ ID NO: RSV065GTCCATTGTTGGATTTAATC 40 RSV093 CAAGATAAGAGTGTACAATACTGTC 41 RSV098TGGTAGTTCTCTTCTGGCTCG 46 RSV105 GTTGGATTGAGAGACACTT 42 RSV112CCCAGTGAATTTATGATTAG 44 RSV113 AGTATTAGGCAATGCTGC 43 RSV114ATCCCCAAGTCATTGTTCA 47 RSV160 AATTGGATCCATGCACACAACCCACAATGA 45

Example 8 Method to Produce Recombinant ΔG+G-RSV Isolate X Virus

ΔG-RSV isolate X virus, derived from transfected Vero cells, waspassaged several times to obtain titres of at least 10⁵ pfu/ml(plaqrsv045ue forming units per ml). Different moi's of this virus werethen used to infect the Vero cell line producing RSV-G protein. Theresulting ΔG+G-RSV isolate X was harvested from the medium and/or fromthe cells and analysed for the presence of the G protein in the virionsby immunodetection techniques. Infectivity titres were determined onVero or Hep-2 cells, and the integrity of the ΔG-genome was determinedusing RT-PCR on viral RNA extracted from cells infected with ΔG+G-RSVisolate X virus. Virus was stored at −135° C. in 25% or 40% sucrose.

Example 9 Method to Protect in a Cotton Rat Animal Model Against RSVInfection and RSV-Induced Pathology By ΔG-RSV Isolate X Immunization

Protection experiments were performed in cotton rats (Sigmodon hispidus,5-6 weeks old, 4-6 animals per group and both sexes), in initialexperiments, this animal was shown to be sensitive to RSV infection andto exhibit severe vaccine-mediated lung pathology as described byPrince, 2001 and which closely mimics the human situation. Afterintranasal application of RSV lung pathology was characterized byinflammation infiltrate in and around bronchus/bronchioli andhyperplasia of epithelium. A more severe pathology was seen uponintramuscular immunization with formalin-inactivated RSV-A2 followed byan intranasal challenge with RSV-A2. In addition to the above-mentionedpathology, perivascular and peribronchiolar infiltrate and alveolitiswere observed, characteristic for an immune-mediated pathology. Theseobservations were used as “internal” reference for all immunization andchallenge experiments. Infection and immunization of cotton rats withRSV preparations was done intranasally, in both nostrils. Cotton ratlungs were examined for pathology lightmicroscopically and virus titresat different time points post-challenge or post-infection/immunizationwere determined on Vero cells using serial dilutions of lung homogenateswith RSV specific ELISA to yield CCID₅₀ titres and immunostaining usingRSV specific abs to yield pfu titres. After immunization twice withΔG-RSV isolate X cotton rats were fully protected against infection andpathology caused by RSV isolate X in the lungs. The results from severalexperiments are summarized in Table IV.

TABLE IV infection lung pathology day with: t¹ V² 5 post infection lungt³ ΔG-RSV isolate X 5 100 yes, moderate below detection RSV-A2 5 100yes, strong 2*5 RSV isolate X 5 100 yes, strong 4*5 immunizationchallenge lung pathology day day 0 and 21 t¹ V² day 42 t¹ V² 5 postchallenge lung t³ 2x ΔG-RSV isolate X 5 100 RSV isolate X 5 100 no belowdetection mock 100 RSV isolate X 5 100 yes, strong 5 ¹virus titres inlogs pfu/ml ²volume in μl per animal, which is half this volume in eachnostril ³virus titres in logs per gram lung, detection limit is 10²CCID₅₀

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1-19. (canceled)
 20. A method for producing pneumoviral virions, thevirions comprising a viral genome that has a mutation in a gene codingfor a protein that is essential for (in vivo) infectivity of thepneumovirus, wherein the mutation causes a virus produced from only theviral genome to lack infectivity, and wherein the virion comprises theprotein in a form and in an amount that is required for infectivity ofthe virion, the method comprising the steps of; (a) infecting a cultureof a first host cell with a pneumovirus comprising a viral genome thathas the mutation, whereby the host cell comprises an expression vectorwhich directs expression in the host cell of the protein in a form andin an amount that is required for infectivity of the virion; and, (b)recovering of the virions from the infected host cell culture.
 21. Themethod according to claim 20, wherein the pneumovirus that is used toinfect the culture of a first host cell culture, is produced by themethod comprising the steps of: (a) providing to a second host cell oneor more expression vectors which direct expression in the host cell of:i) a viral genomic RNA that has a mutation in a gene coding for aprotein that is essential for (in vivo) infectivity of the pneumovirus,whereby the mutation causes a virus produced from only the viral genometo lack infectivity; ii) a pneumoviral polymerase enzyme complex andoptionally one or more further viral proteins; and, (b) culturing thesecond host cell whereby the virions are produced.
 22. The methodaccording to claim 21, further comprising amplifying the virionsproduced by the second host cell by one or more further cellularinfection steps employing host cells which are the same or differentfrom the second host cell.
 23. The method according to claim 21, whereinthe viral genomic RNA is transcribed from a viral DNA copy that is underthe control of a bacteriophage DNA-dependent RNA polymerase promoter andwherein the host cell is provided with an expression vector whichdirects expression in the host cell of the bacteriophage DNA- dependentRNA polymerase.
 24. The method according to claim 23, wherein thebacteriophage DNA-dependent RNA polymerase is the T7, T3 or SP6polymerase.
 25. The method according to claim 21, wherein thepneumoviral polymerase enzyme complex at least includes the L, P, Nproteins.
 26. The method according to claim 21, whereby one or morefurther viral proteins is a pneumoviral matrix membrane protein.
 27. Themethod according to claim 26, wherein the a pneumoviral matrix membraneprotein is the M2-1 protein.
 28. The method according to claim 20,wherein the pneumovirus is a Respiratory Syncytial Virus.
 29. The methodaccording to claim 20, wherein the gene coding for the protein that isessential for infectivity is a gene coding for a G attachment protein.